The invention relates to polyol-IFN-xcex2 conjugates wherein a polyol unit is covalently bound to Cys17. Further objects of the present invention are the process for their site-specific production as well as their use in the therapy, prognosis or diagnosis of bacterial infections, viral infections, autoimmune diseases and inflammatory diseases. The present invention further relates to a method for the stepwise attachment of two or more PEG moieties to a polypeptide.
Human fibroblast interferon (IFN-xcex2) has antiviral activity and can also stimulate natural killer cells against neoplastic cells. It is a polypeptide of about 20,000 Da induced by viruses and double-stranded RNAs. From the nucleotide sequence of the gene for fibroblast interferon, cloned by recombinant DNA technology, Derynk et al. (Nature, 285:542-547, 1980) deduced the complete amino acid sequence of the protein. It is 166 amino acid long.
Shepard et al. (Nature, 294:563-565, 1981) described a mutation at base 842 (Cysxe2x86x92Tyr at position 141) that abolished its anti-viral activity, and a variant clone with a deletion of nucleotides 1119-1121.
Mark et al. (Proc. Natl. Acad. Sci. U.S.A., 81(18):5662-5666, 1984) inserted an artificial mutation by replacing base 469 (T) with (A) causing an amino acid switch from Cysxe2x86x92Ser at position 17. The resulting IFN-xcex2 was reported to be as active as the xe2x80x98nativexe2x80x99 IFN-62  and stable during long-term storage (xe2x88x9270xc2x0 C.).
Covalent attachment of the hydrophilic polymer polyethylene glycol, (PEG), also known as polyethylene oxide, (PEO), to molecules has important applications in biotechnology and medicine. In its most common form, PEG is a linear polymer having hydroxyl groups at each terminus:
HOxe2x80x94CH2xe2x80x94CH2O(CH2CH2O)nCH2CH2xe2x80x94OH
This formula can be represented in brief as HO-PEG-OH, where it is meant that -PEG- represents the polymer backbone without the terminal groups:
xe2x80x9c-PEG-xe2x80x9d means xe2x80x9cxe2x80x94CH2CH2O(CH2CH2O)nCH2CH2xe2x80x94
PEG is commonly used as methoxy-PEG-OH, (m-PEG), in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to chemical modification.
CH3Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH
Branched PEGs are also in common use. The branched PEGs can be represented as R(-PEG-OH)m in which R represents a central core moiety such as pentaerythritol or glycerol, and m represents the number of branching arms. The number of branching arms (m) can range from three to a hundred or more. The hydroxyl groups are subject to chemical modification.
Another branched form, such as that described in PCT patent application WO 96/21469, has a single terminus that is subject to chemical modification. This type of PEG can be represented as (CH3O-PEG-)pRxe2x80x94X, whereby p equals 2 or 3, R represents a central core such as lysine or glycerol, and X represents a functional group such as carboxyl that is subject to chemical activation. Yet another branched form, the xe2x80x9cpendant PEGxe2x80x9d, has reactive groups, such as carboxyl, along the PEG backbone rather than at the end of PEG chains.
In addition to these forms of PEG, the polymer can also be prepared with weak or degradable linkages in the backbone. For example, Harris has shown in U.S. patent application Ser. No. 06/026,716 that PEG can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis. This hydrolysis results in cleavage of the polymer into fragments of lower molecular weight, according to the reaction scheme:
xe2x80x83-PEG-CO2-PEG-+H2Oxe2x86x92-PEG-CO2H+HO-PEG-
According to the present invention, the term polyethylene glycol or PEG is meant to comprise all the above described derivatives.
The copolymers of ethylene oxide and propylene oxide are closely related to PEG in their chemistry, and they can be used instead of PEG in many of its applications. They have the following general formula:
HOxe2x80x94CH2CHRO(CH2CHRO)nCH2CHRxe2x80x94OH
wherein R is H or CH3. 
PEG is a useful polymer having the property of high water solubility as well as high solubility in many organic solvents. PEG is also non-toxic and non-immunogenic. When PEG is chemically attached (PEGylation) to a water insoluble compound, the resulting conjugate generally is water soluble, as well as soluble in many organic solvents.
PEG-protein conjugates are currently being used in protein replacement therapies and for other therapeutic uses. For example, PEGylated adenosine deaminase (ADAGEN(copyright)) is being used to treat severe combined immunodeficiency disease (SCIDS), PEGylated L-asparaginase (ONCAPSPAR(copyright)) is being used to treat acute lymphoblastic leukemia (ALL), and PEGylated interferon-xcex1 (INTRON(R) A) is in Phase III trials for treating hepatitis C.
For a general review of PEG-protein conjugates with clinical efficacy see N. L. Burnham, Am. J. Hosp. Pharm., 15:210-218, 1994.
A variety of methods have been developed to PEGylate proteins. Attaching PEG to reactive groups found on the protein is typically done utilizing electrophilically activated PEG derivatives. Attaching PEG to the xcex1- and xcex5-amino groups found on lysine residues and the N-terminus results in a conjugate consisting of a mixture of products.
Generally, such conjugates consist of a population of the several PEG molecules attached per protein molecule (xe2x80x9cPEGmersxe2x80x9d) ranging from zero to the number of amino groups in the protein. For a protein molecule that has been singly modified, the PEG unit may be attached at a number of different amine sites.
This type of non-specific PEGylation has resulted in a number of conjugates that become almost inactive. Reduction of activity is typically caused by shielding the protein""s active binding domain as is the case with many cytokines and antibodies. For example, Katre et al. in U.S. Pat. Nos. 4,766,106 and 4,917,888 describe the PEGylation of IFN-xcex2 and IL-2 with a large excess of methoxy-polyethylene glycolyl N-succinimidyl gluterate and methoxy-polyethylene glycolyl N-succinimidyl succinate. Both proteins were produced in microbial host cells, which allowed the site-specific mutation of the free cysteine to a serine. The mutation was necessary in microbial expression of IFN-xcex2 to facilitate protein folding. In particular, the IFN-xcex2 used in these experiments is the commercial product Betaseron(copyright), in which Cys17 residue is replaced with a serine. Additionally, the absence of glycosylation reduced its solubility in aqueous solution. Non-specific PEGylation resulted in increased solubility, but a major problem was the reduced level of activity and yield.
European Patent Application EP 593 868, entitled PEG-Interferon Conjugates, describes the preparation of PEG-IFN-xcex1 conjugates. However, the PEGylation reaction is not site-specific, and therefore a mixture of positional isomers of PEG-IFN-xcex1 conjugates are obtained (see also Monkarsh et al., ACS Symp. Ser., 680:207-216, 1997).
Kinstler et al. in European Patent Application EP 675 201 demonstrated the selective modification of the N-terminal residue of megakaryocyte growth and development factor (MGDF) with mPEG-propionaldehyde. This allowed for reproducible PEGylation and pharmacokinetics from lot to lot. Gilbert et al. in U.S. Pat. No. 5,711,944 demonstrated that PEGylation of IFN-xcex1 with an optimal level of activity could be produced. In this instance a laborious purification step was needed to obtain the optimal conjugate.
The majority of cytokines, as well as other proteins, do not possess a specific PEG attachment site and, apart from the examples mentioned above, it is very likely that some of the isomers produced through the PEGylation reaction be partially or totally inactive, thus causing a loss of activity of the final mixture.
Site-specific mono-PEGylation is thus a desirable goal in the preparation of such protein conjugates.
Woghiren et al. in Bioconjucate Chem., 4(5):314-318, 1993, synthesized a thiol-selective PEG derivative for such a site-specific PEGylation. A stable thiol-protected PEG derivative in the form of an parapyridyl disulfide reactive group was shown to specifically conjugate to the free cysteine in the protein, papain. The newly formed disulfide bond between papain and PEG could be cleaved under mild reducing conditions to regenerate the native protein.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicants at the time of filing and does not constitute an admission as to the correctness of such a statement.
In the present invention, polyol-IFN-xcex2 conjugates, and particularly PEG-IFN-xcex2 conjugates, are provided wherein a polyol unit is covalently bound to Cys17. The specific conjugation is obtained by allowing a thiol-reactive polyol agent to react with the Cys17 residue in IFN-xcex2. Such conjugates are expected to show increased effectiveness in vivo. The aim is to obtain increased solubility at neutral pH, increased stability (decreased aggregation), decreased immunogenicity, and no loss of activity with respect to xe2x80x98nativexe2x80x99 IFN-xcex2. The results of such conjugation would decrease the number of doses for an intended effect, simplify and stabilize the formulation of a pharmaceutical composition, and possibly increase the long-term efficacy.
The present invention further provides a method for the stepwise attachment of PEG moieties in series to a polypeptide.